Genetic and epigenetic information is important in understanding a number of diseases important to humans, including diseases such as Alzheimer's disease, schizophrenia, diabetes, atherosclerosis, Parkinson's disease, and cancer. Tools that enhance the ability to understand the complex genetic and epigenetic machinery relating to various diseases and conditions are crucial in developing treatments for these diseases.
Epigenetics is the study of modifications in gene expression caused by chemical modification of the chromosome with no changes in the underlying DNA sequence. Among the numerous chemical modifications, methylation of DNA at 5-cytosine is one of the most widely studied mechanisms influencing gene regulation. One of the effects of DNA methylation is to physically impede the binding of transcription factors to their recognition sites, and another is to bind methyl-CpG-binding domain (MBD) proteins involved in the modification of chromatin. In recent years, the field of epigenetics has become one of the most rapidly growing branches of molecular biology with increasing effort devoted to efficiently characterizing the human epigenome. The number of diseases suspected of being influenced by DNA methylation is growing and includes Alzheimer's disease, schizophrenia, diabetes, atherosclerosis, Parkinson's disease, cancer, among others (Schumacher et al., 2006, 310, 81-115). Interrogation of DNA methylation patterns in regulatory regions such as CpG islands has become an important tool for medical diagnostics and understanding of gene regulation. DNA methylation detection could serve, in particular, as a tool in cancer diagnosis and monitoring of treatment.
Current methods for methylation assessment and analysis include sequencing after bisulfate conversion (Cokus et al., Nature 2008, 452, 215), methylation-specific polymerase chain reaction (PCR) (Fraga et al., Biotechniques 2002, 33, 632), methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) (Gonzalgo et al., Nat. Protoc. 2007, 2, 1931), combined bisulfate restriction analysis (COBRA) (Xiong et al., Nucleic Acids Res. 1997, 25, 2532), methylated DNA immunoprecipitation (Weber et al., Nat. Genet. 2005, 37, 853), restriction landmark genome scanning (Ando et al., Nat. Protoc. 2006, 1, 2774), hybridization arrays (Bibikova et al., Genome Res. 2006, 16, 383; Nautiyal et al., Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 12587), among others. Efficient individual molecule analysis could reveal information lost in current ensemble methods of bulk material analysis and allow investigation of single cells. While the benefits of single molecule analyses are known and research is underway on new such methods, they are not yet available for general use. Current single molecule methods being studied include fluorescent detection of methylation in nanofluidic systems (Cipriany et al., Anal. Chem. 2010, 82, 2480-2487; Lim et al., Biomicrofluidics 2011, 5, 034106) and observation of rate dependence of polymerase activity as influenced by methylation (Flusberg et al., Nat. Meth. 2010, 7, 461-465). However, none of these methods is yet in general use in epigenetic analyses and the need for robust methods still exists.
Chromatin consists of repeating nucleosome units that contain two pairs of four types of histone proteins (H2A, H2B, H3 and H4) forming an octamer or eight-unit histone core, that is wrapped 1.65 turns by a 147-base length of DNA controlling access to the underlying sequence. The composition, modification and structure of chromatin plays a crucial role in gene expression. In mammalian organisms, epigenetic gene regulation functions through methylation of CpG dinucleotides and remodeling of chromatin structure through post-translational histone modifications such as acetylation, methylation, ubiquitylation, poly(ADP)ribosylation, and phosphorylation that can affect the biophysical properties and signaling of regulatory factors within the chromatin template. In the study of a variety of diseases, there is an active effort to map genome-wide genetic and epigenetic patterns across cell types, the latter in response to various environmental influences for the understanding of gene regulation and for medical diagnostics (Barski et al., Cell. 2007, 129, 823-837; Bernstein et al., Cell. 2007, 128, 669-681; Bianchi-Frias et al., PLoS Biology. 2004, 2, 975-990). Interrogation of chromatin modifications could serve as a tool for diagnosis and monitoring the effectiveness of treatment. In non-mammalian organisms such as drosophila, chromatin profiling would provide a means of identifying transcriptional target genes and a global view of cofactor recruitment during development (Buck et al., Genomics. 2004, 83, 349-360).
High resolution imaging methods, facilitated by stretching and immobilization of chromatin, may provide a direct approach to identifying epigenetic modifications throughout the genome. While the majority of current studies investigate large cell populations through coupling chromatin immunoprecipitation protocols (ChIP) with either hybridization arrays or sequencing (Heng et al., PNAS. 1992, 89, 9509-9513) or through Fluorescence In-Situ Hybridization (FISH)-like visualization at the gross levels of chromosomal superstructure (Smith et al., Science. 1992, 258, 1122-1126), few studies are aimed towards studying chromatin at the fine scale. The concept of stretching nucleic acids for analysis has been largely exploited for the study of bare DNA. Current techniques for DNA stretching comprise end-tethering, usually in combination with optical or magnetic tweezers (Smith et al., Science. 1996, 271, 795-799; Cerf et al., Anal. Chem. 2011, 83, 8073-8077), stretching on polydimethylsiloxane (PDMS) stamps (Gad et al., J. Biomol. Struct. Dyn. 2003, 2, 387-393; Björk et al., Small, 2006, 8-9, 1068-1074; Nakao et al., Nano Lett. 2003, 3, 1391-1394; Nakao et al., JACS, 2003, 125, 7162-7163; Guan et al., PNAS, 2005, 102, 18321-18325; Bensimon et al., Science. 1994, 265, 2096-2098), adsorption onto a modified surface under flow (Greene et al., Methods Enzymol. 2010, 472, 293-315; Perkins et al., Science. 1995, 268, 83-87; Tegenfeldt et al., Phys. Rev. Lett. 2001, 86, 1378-1381), shear flow (Smith et al., Science. 1999, 283, 1724-1727; Wang et al., “Microfluidic Extraction and Stretching of Chromosomal DNA from Single Cell Nuclei for DNA Fluorescence In Situ Hybridization,” Biomed. Microdevices. 2012, in press; Tegenfeldt et al., PNAS. 2004, 101, 10979-10983), and nanoconfinement (Reccius et al., Biophys. J. 2008, 95, 273-286; Cui et al., PNAS. 2000, 97, 127-132). While these techniques should be transposable to chromatin, it is noticeable that chromatin stretching is less studied, except for end-tethered stretching (Bennink et al., Nat. Struct. Biol. 2001, 8, 606-610; Leuba et al., PNAS. 2003, 100, 495-500; Brower-Toland et al., PNAS. 2002, 99, 1960-1965; Gorman et al., Nat. Struct. Mol. Biol. 2010, 17, 932-938; Bancaud et al., Nat. Struct. Mol. Biol. 2006, 13, 444-450; Streng et al., Lab on a Chip. 2009, 9, 2772-2774) and a few recent nanoconfinement studies (Cipriany et al., Anal. Chem. 2010, 82, 2480-2487; Ersfeld, K., Meth. in Mol. Biol. 2004, 270, 395-402). The most popular stretching technique for FISH-like genetic or epigenetic analysis relies on cell shearing after fixation, the latter leading to poor molecule to molecule repeatability (Smith et al., Science. 1992, 258, 1122-1126; Sims et al., J. Biol. Chem. 2006, 281, 12760-12766). As an example, chromatin fibers from drosophila and human cells have been spread by direct lysis onto charged microscope slides to show the organization of covalently modified histones in silent chromatin sequences (Blower et al., Dev. Cell. 2002, 2, 319-330), DNA replication timing in centromeric regions (Lam et al., PNAS. 2006, 103, 4186-4191; Sullivan et al., Nat. Struct. Mol. Biol. 2004, 11, 1076-1083; Cohen et al., Epigenetics & Chromatin. 2009, 2, 6), and to study distribution of GINS complex (Daban, J. R., Micron. 2011, 42, 733-750). Other studies involve stretching chromatin fibers to establish a mapping between spatial location and genomic location (Smith et al., Science. 1992, 258, 1122-1126; Greene et al., Methods Enzymol. 2010, 472, 293-315) or for morphological studies using transmission electron microscopy and atomic force microscopy (Lyubchenko et al., Methods. 2009, 47, 206-213). In recent years, progress has been made in employing new technologies with the potential to bridge the gap between static structural features and dynamic physiological processes in the study of biomolecules, while increasing spatial resolution (Kobayashi et al., Ultramicroscopy. 2007, 107, 184-190; Ando et al., Pflugers Arch. 2008, 456, 211-225; Cerf et al., J. Mater. Res. 2010, 26, 336-346). Yet, to our knowledge, no method has been demonstrated that allows the controllable stretching and isolation of canonical and native chromatin molecules for high-throughput analyses while being compatible with high-resolution imaging techniques.
The present invention is directed to overcoming these and other deficiencies in the art.